In this competing renewal a team of interdisciplinary investigators, who have an established track record of collaboration, will examine the cellular and molecular mechanisms of lipid-induced hepatic insulin resistance. Recent studies in both humans and rodent models of non alcoholic fatty liver disease (NAFLD) have led to a unifying diacylglycerol-hypothesis (DAG-hypothesis) for lipid-induced hepatic insulin resistance where accumulation of intracellular DAG activates protein kinase C5 (PKC5), directly leading to inhibition of insulin- stimulated insulin receptor kinase (IRK) activity. Specific Aim 1 wil explore the molecular mechanism by which activation of PKC5 leads to inhibition of IRK activity. This aim wil examine the hypothesis that PKC5 activation promotes increased threonine phosphorylation of IRK, which in turn blocks insulin-stimulated IRK activity, utilizing state-of-the-art LC-MS/MS methods to identify novel PKC5-induced IRK phosphothreonine sites. Specific Aim 2 wil further examine the DAG-hypothesis by examining the effect of a potential novel therapeutic target (INDY, acronym for I am Not Dead, Yet) on lipid-induced hepatic insulin resistance in awake whole body INDY knockout mice as well as awake rats with liver specific knockdown expression of INDY utilizing mRNA selective antisense oligonucleotides. INDY encodes a non-electrogenic dicarboxylate and citrate transporter and has been shown to promote longevity in a manner akin to caloric restriction in flies, but its role in mammals is unknown. This aim builds on our strong preliminary data demonstrating that INDY knockout mice exhibit increased whole body energy expenditure and are protected from lipid-induced whole body insulin resistance. Specific Aim 3 wil examine the mechanism by which knockdown of INDY in liver leads to increased whole body energy expenditure by employing a novel state-of-the-art 13C/31P NMR method to measure in vivo rates of hepatic fat oxidation, hepatic pyruvate oxidation, hepatic mitochondrial TCA flux, hepatic mitochondrial ATP synthesis and hepatic mitochondrial energy coupling in awake rats for the first time. This aim will examine the hypothesis that decreased hepatic expression of INDY will result in increased rates of hepatic mitochondrial TCA flux, increased rates of hepatic fatty acid oxidation, and decreased hepatic mitochondrial energy coupling, which results in lower hepatic DAG content and protection from lipid-induced hepatic insulin resistance. It is anticipated that the results from these studies will provide important new insights into the cellular mechanisms of NAFLD associated hepatic insulin resistance as well as the identification of a novel therapeutic target for the treatment of NALFD and T2D. Furthermore, once validated in these animal experiments, these 13C/31P NMR methods will be translated to man to directly assess liver specific rates of hepatic mitochondrial TCA flux, mitochondrial ATP synthesis and hepatic mitochondrial energy coupling in humans for the first time.